The portal theory supported by venous drainage-selective fat transplantation

Abstract

Objective: The "portal hypothesis" proposes that the liver is directly exposed to free fatty acids and cytokines increasingly released from visceral fat tissue into the portal vein of obese subjects, thus rendering visceral fat accumulation particularly hazardous for the development of hepatic insulin resistance and type 2 diabetes. In the present study, we used a fat transplantation paradigm to (artificially) increase intra-abdominal fat mass to test the hypothesis that venous drainage of fat tissue determines its impact on glucose homeostasis.

Research design and methods: Epididymal fat pads of C57Bl6/J donor mice were transplanted into littermates, either to the parietal peritoneum (caval/systemic venous drainage) or, by using a novel approach, to the mesenterium, which confers portal venous drainage.

Results: Only mice receiving the portal drained fat transplant developed impaired glucose tolerance and hepatic insulin resistance. mRNA expression of proinflammatory cytokines was increased in both portally and systemically transplanted fat pads. However, portal vein (but not systemic) plasma levels of interleukin (IL)-6 were elevated only in mice receiving a portal fat transplant. Intriguingly, mice receiving portal drained transplants from IL-6 knockout mice showed normal glucose tolerance.

Conclusions: These results demonstrate that the metabolic fate of intra-abdominal fat tissue transplantation is determined by the delivery of inflammatory cytokines to the liver specifically via the portal system, providing direct evidence in support of the portal hypothesis.

FIG. 1.

Transplantation was performed at 6 weeks of age. Both epididymal fat pads were removed from a donor mouse and stitched to the mesenterium (pTx) of a littermate recipient. A: Six weeks after transplantation, the abdominal cavity was opened and the transplanted fat pad was visualized. B: To confirm portal drainage of transplanted fat pads, Evans blue dye (5 mg/ml) was injected into the portal vein. Arrow: portal transplanted fat pad. C: Intraperitoneal glucose tolerance test (2 g glucose/kg body weight) was performed 4 weeks after surgical procedure in mice receiving portal drained transplants (pTx; ○), in mice receiving caval drained transplants (cTx; ), or in sham-operated mice (●) after an overnight fast. Results are expressed as mean blood glucose concentration (left panel) or area under curve (AUC) (right panel) ± SEM of 5 (pTx), 16 (cTx), or 23 (sham) animals. **P < 0.01, #P < 0.001 (left panel: Student t test [pTx vs. sham]; right panel: ANOVA). D: Both epididymal fat pads were removed from a donor mouse. One was stitched to the peritoneum (caval drained; cTx) and the other to the mesenterium (portal drained; pTx) of a littermate. Intraperitoneal glucose tolerance test (2 g glucose/kg body wt) was performed 4 weeks after surgical procedure in mice receiving a double transplant (cTx + pTx; ▵) or in sham-operated mice (●) after an overnight fast. Results are expressed as mean blood glucose concentration ± SEM of three to eight animals. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

A: Representative hematoxylin- and eosin-stained histological sections of endogenous epididymal, endogenous mesenteric, or portal-transplanted fat pads from sham-operated and pTx mice are shown 5 weeks after surgery. Adipocyte cell perimeter was measured using ImageJ. B: Up to 100 cells for each fat pad were analyzed, and the results represent the mean ± SEM of three or four different mice. Black bar: sham-operated mice; white bar: mice receiving a portal fat transplant. C: Histological sections of endogenous epididymal and portal-transplanted tissue were immunostained with mac2 antibody. Mac2-positive cells were counted in seven randomly selected fields per section (about 90 cells) and expressed relative to total cell nucleus content. Results represent the mean ± SEM of three to five different mice. All values are expressed relative to Mac2-positive cells of epididymal fat pad of sham-operated mice.

FIG. 3.

A and B: Hyperinsulinemic-euglycemic clamps were performed 5 weeks after transplantation. A: Mean glucose infusion rate (see research design and methods). B: Endogenous glucose production (EGP; left panel) as well as insulin-stimulated glucose disposal rate (IS-GDR; right panel) were calculated in the basal period and in response to insulin infusion (18 mU/kg*min) during the clamp study. Results are the mean ± SEM of five to six animals. *P < 0.05 (Student t test). C: Insulin (1 U/kg) was injected intraperitoneally in overnight fasted mice 5 weeks after transplantation. Liver and skeletal muscle samples were harvested 15 min later, and phosphorylation of Akt at S473 was determined by Western blotting. A representative blot is shown in the upper panel. Results are the mean ± SEM of three to four animals. **P < 0.01 (Student t test). D: Blood samples were obtained as indicated, and insulin was determined as described in research design and methods. Results are expressed as mean plasma insulin concentration ± SEM of five to six animals.

FIG. 4.

A: Left panel: Hematoxylin- and eosin-stained liver sections from sham-operated and pTx mice. Right panel: Total liver lipids were determined and expressed relative to liver weight. Results represent the mean ± SEM of four to six mice of each group. B: mRNA expression of indicated markers in liver tissue of sham-operated and pTx mice was analyzed. Results are the mean ± SEM of five mice per group, expressed relative to sham-operated mice and normalized to expression of 18S. C: FFA levels were determined in systemic and portal plasma samples of mice receiving a portal drained fat transplant (pTx) and in sham-operated mice after 3 h of fasting. Results are the mean ± SEM of five to six animals.

FIG. 5.

A: Five weeks after transplantation, endogenous epididymal fat pads (endo), the portal-drained transplant (pTx), and the caval-drained transplant (cTx) were collected and real-time PCR was performed. Results are the mean ± SEM of two to seven animals. **P < 0.01 pTx vs. cTx (ANOVA). B: IL-6 was measured in systemic and portal plasma samples of mice receiving a portal-drained fat transplant (pTx) and in sham-operated mice. Results are the mean ± SEM of 8–10 animals. C: Five weeks after transplantation, liver tissue was collected and rtPCR was performed. Results are the mean ± SEM of four to five animals.

FIG. 6.

Both epididymal fat pads were removed from an IL-6 knockout mouse (donor) and stitched to the mesenterium of a wild-type mouse (recipient; pTx-IL6KO). A: Intraperitoneal glucose tolerance test (2 g glucose/kg body wt) was performed 4 weeks after surgical procedure in mice receiving portal transplants (pTx-IL6KO; ♢) or in sham-operated mice (●). B: Insulin (1 U/kg) was injected intraperitoneally in overnight fasted mice 5 weeks after transplantation. Liver samples were harvested 15 min later, and phosphorylation of Akt at S473 was determined by Western blotting. A representative blot is shown in the upper panel. Results are the mean ± SEM of three animals. C: Five weeks after transplantation, liver tissue was collected and real-time PCR was performed. Results are the mean ± SEM of two to three animals.